Non-mammalian transgenic animal model for cellular proliferative diseases

ABSTRACT

Non-mammalian, transgenic animals, e.g. flies, that exhibit neoplastic phenotypes, i.e. spontaneously produce metastatic tumors, are provided. Also provided are methods of using the subject transgenic non-mammalian animals to identify compounds having activity with respect to cellular proliferative, and particularly neoplastic, diseases. Finally, kits for screening compounds for anti-neoplastic activity are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of U.S. patent applicationSer. No. 09/472,661, filed Dec. 27, 1999, which claims priority to thefiling date of the U.S. Provisional Patent Application Ser. No.60/147,433 filed Aug. 4, 1999, the disclosure of which is hereinincorporated by reference.

FIELD OF THE INVENTION

The field of this invention is animal models of cellular proliferativediseases.

BACKGROUND OF THE INVENTION

Cancer remains one of the leading causes of death in the United States.Clinically, a broad variety of medical approaches, including surgery,radiation therapy and chemotherapeutic drug therapy are currently beingused in the treatment of human cancer (see the textbook CANCER:Principles & Practice of Oncology, 2d Edition, De Vita et al., eds., J.B. Lippincott Company, Philadelphia, Pa., 1985). With respect tochemotherapeutic drug therapy approaches, a broad variety of differenttypes of active agents have been identified that exhibit anti-cellularproliferative activity, and some of these agents currently find use inthe treatment of cancer and related cellular proliferative diseases.Agents which have been found to exhibit anti-cellular proliferativeactivity include: cytostatics, plant alkaloids, antibiotics, etc.However, despite the large number of compounds that have been identifiedto date, no ideal drug therapeutic has yet been discovered.

Critical steps in the identification and development of new therapeuticagents are: (a) generation of candidate agents; and (b) screening of thecandidate agents for efficacy and safety. With the advent ofcombinatorial chemistry protocols, large numbers of potential compounds,known as libraries, can be rapidly generated. Such libraries serve ascollections of potential therapeutic agents. Following generation of alibrary of potential therapeutic agents, the library must be screened toidentity the promising candidates.

For screening purposes, a number of in vitro high throughput screeningprotocols have been developed. However, these in vitro screening assaysmust be followed by in vivo screening assays. Since it is undesirable toimmediately screen compounds that show promise from in vitro assays inhumans, an important step in the identification of therapeutic agentsfor such cellular proliferative diseases is the screening of potentialtherapeutic compounds in non-human animal models. As such, non-humananimal models of cancer and other cellular proliferative diseases playan important role in the discovery of therapeutic agents for suchdiseases.

One type of non-human animal model that can be used for screeningpurposes to identify therapeutic agents for use in treating cancer andother cellular proliferative diseases is a non-human mammalian model,e.g. mice, etc. However, mice are expensive, have a slow reproductiontime, and generate small numbers of offspring. As such, they are lessthan ideal for many high throughput screening assays.

Accordingly, there is a need for additional animal models of cellularproliferative, e.g. neoplastic, diseases. Of particular interest wouldbe the development of an animal model having a relatively short lifespan and a rapid reproduction cycle characterized by the production oflarge numbers of offspring. Preferably, such an animal model should alsobe relatively simple and economic to maintain.

Relevant Literature

Of interest is: Woodhouse et al., Dev. Genes. Evol. (1998) 207:542-550.Methods of preparing transgenic Drosophila melanogaster are disclosedin: Spradling, A. C., and Rubin, G. M. (1982). Science 218, 341-347;Brand & Perrimon, Development (1993) 118: 401-415; and Phelps & Brand,Methods (April 1998) 14:367-379. See also, Spradling A C, P ElementMediated Transformation in Drosophila: A Practical Approach (ed. D. D.Roberts, IRL Press, Oxford)(1986) pp 175-179.

SUMMARY OF THE INVENTION

Transgenic non-mammalian animals, e.g. flies, that have a neoplasticphenotype, i.e. that spontaneously develop metastatic tumors, areprovided. The subject transgenic animals are characterized in that theyhave a v-myb transgene stably integrated into their genome that isexpressed in a manner that gives rise to the neoplastic phenotype. Alsoprovided are methods of screening compounds for activity with respect tocellular proliferative disease conditions, particularly compounds havingtherapeutic activity with respect to neoplastic disease conditions.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A provides a computer generated representation of a wild typelarva; FIG. 1B provides a computer generated representation of atumorous larva according to the subject invention; FIG. 1C also providesa copmuter generated representation of a tumorous larva according to thesubject invention.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Non-mammalian transgenic animals, particularly insects, e.g. flies, thathave neoplastic phenotypes are provided. The subject transgenic animalsare characterized in that they spontaneously develop metastatic tumors.The subject animals comprise a v-myb transgene that is expressed in amanner that gives rise to the neoplastic phenotype. Also provided aremethods of using the subject non-mammalian transgenic animals to screenfor compounds having activity with respect to cellular proliferativediseases, particularly compounds that are therapeutic for neoplasticdiseases. In further describing the subject invention, the transgenicanimals and methods for their production will be detailed first,followed by a discussion of the screening methods of the subjectinvention.

Before the subject invention is described further, it is to beunderstood that the invention is not limited to the particularembodiments of the invention described below, as variations of theparticular embodiments may be made and still fall within the scope ofthe appended claims. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting. Instead, the scope of the present inventionwill be established by the appended claims.

In this specification and the appended claims, the singular forms ●a, ●●an, “and ●the” include plural reference unless the context clearlydictates otherwise. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood to one of ordinary skill in the art to which this inventionbelongs.

Animal Models

The invention provides non-mammalian animal models that exhibit aneoplastic phenotype, where the phenotype results from the expression ofa transgene in the proper spatial manner, as described in greater detailbelow. By neoplastic phenotype is meant that the subject transgenicnon-mammalian animal models spontaneously develop metastatic tumors atsome point during their life-span. Thus, a particular multicellularorganism is a transgenic animal according to the subject invention nomatter which stage of development it is at, so long as the animalspontaneously develops metastatic tumors (at least one) at some pointduring its lifetime. Transgenic animals having the neoplastic phenotypiccharacteristics of the subject invention are readily identified, as theyhave the following phenotypic characteristics: (1) development of darkcolored, dense clusters of dividing cells at some point during theirlifetime, where the clusters (or a portion thereof) can be dissectedfrom the transgenic animal and grown in culture, where the size of theculture grown cluster of cells doubles in about 4 to 10 days, usuallyabout 5 to 7 days; and (2) transplantation of the tumors from thesubject transgenic animals into normal recipients, e.g. larva, tends toinduce death.

The transgenic animals of the subject invention are non-mammaliantransgenic animals. Of particular interest are invertebrate transgenicanimals, particularly members of the phylum arthropoda, and moreparticularly members of the class insecta. Of particular interest inmany embodiments are transgenic flies. In many preferred embodiments,the transgenic flies are members of the family Drosophilidae, where thetransgenic animal is often a Drosophila melanogaster. The subjectinvention is now further described in terms of transgenic flies.

A critical feature of the subject transgenic animals is that the animalsharbor a stably integrated transgene that is spatially expressed in amanner sufficient to result in the desired neoplastic phenotype. Theterm “transgene” is used herein to describe genetic material which hasbeen or is about to be artificially inserted into the genome of a cell.With regards to spatial expression of the transgene, expression isgenerally limited to the arinioserosa and peripheral nervous system.

The transgene encodes a product that, when expressed in the appropriatespatial manner, gives rise to the neoplastic phenotype. As mentionedabove, the transgene is a v-myb gene, where the v-myb gene may be thenaturally occurring v-myb gene or a gene that encodes a product which,though not identical to the product encoded by the naturally occurringv-myb gene, nonetheless gives rise to the neoplastic phenotype. Thus,the transgene may comprise a nucleic acid having the sequence of thenaturally occurring v-myb gene or a sequence that includes only a v-mybdomain. As such, the transgene at least includes a v-myb domain or asequence that is substantially similar to a domain found in thenaturally occurring v-myb, where substantially similar means a nucleicacid sequence having a sequence identity with a naturally occurringv-myb domain of at least about 50%, usually at least about 60% and moreusually at least about 65%, where sequence identity is determined usingthe BLAST program at default settings (using default settings, i.e.parameters w=4 and T=17). In many embodiments, the transgene comprises anaturally occurring v-myb gene or a sequence substantially similarthereto, where viral myb genes of interest include that induceoncogenesis in vertebrates, such as: v-myb-1151 as described in Fu, S.L. and J. S. Lipsick (1996) J. Vir. 70: 5600-5610; AMV-vmyb, E26 vmyb,dGE vmyb, and 1183 vmyb where each of these is described in Ganter &Lipsick, Adv. Cancer Res. (1999) 76:21-60, and the references disclosedtherein.

The v-myb transgene is stably integrated into the genome of the animalin manner such that its expression is controlled spatially to thedesired cell type. Specifically, the subject transgene is stablyintegrated into the genome of the animal under the control of a promoterthat provides for expression in at least the amnioserosa and/orperipheral nerve system. The transgen-e may be under the control of anyconvenient promoter that provides for this requisite spatial expressionpattern, where the promoter may be endogenous or exogenous, but willgenerally be endogenous. A suitable promoter is the promoter located onthe 4^(th) chromosome in the Drosophila melanogaster genome thatregulates GAL4 expression in stock #3737 (Bloomington, Ind.designation).

The transgene may be integrated into the fly genome in a manner thatprovides for direct or indirect expression activation by the promoter,i.e. in a manner that provides for either cis or trans activation ofgene expression by the promoter. In other words, expression of thetransgene may be mediated directly by the promoter, or through one ormore transactivating agents. Where the transgene is under direct controlof the promoter, i.e. the promoter regulates expression of the transgenein a cis fashion, the transgene is stably integrated into the genome ofthe fly at a site sufficiently proximal to the promoter and in framewith the promoter such that cis regulation by the promoter occurs.

In yet other embodiments where expression of the transgene is indirectlymediated by the endogenous promoter, the promoter controls expression ofthe transgene through one or more transactivating agents, usually onetransactivating agent, i.e. an agent whose expression is directlycontrolled by the promoter and which binds to the region of thetransgene in a manner sufficient to turn on expression of the transgene.Any convenient transactivator may be employed, where the GAL4transactivator system is particularly preferred in many embodiments ofthe subject invention.

In these preferred embodiments of the subject invention in which thetransgenic fly comprises the GAL4 targeted expression system, a GAL4encoding sequence is stably integrated into the genome of the animal ina manner such that it is operatively linked to the endogenous promoterthat provides for expression in the appropriate spatial and temporalmanner. An example of such a fly is fly line #3734 available from theBloomington Stock Center (Bloomington,Ind.)(http://flybase.bio.indiana.edu/). The transgene is stablyintegrated into a different location of the genome, generally a randomlocation in the genome, where the transgene is operatively linked to anupstream activator sequence, i.e. UAS sequence, to which GAL4 binds andturns on expression of the transgene. Transgenic flies having a UAS:GAL4 transactivation system are known to those of skill in the art andare described in Brand & Perrimon, Development (1993) 118: 401-415; andPhelps & Brand, Methods (April 1998) 14:367-379.

Methods of Producing the Subject Transgenic Flies

The subject transgenic flies can be prepared using any convenientprotocol that provides for stable integration of the transgene in to thefly genome in a manner sufficient to provide for the requisite spatialexpression of the transgene, i.e. in mainly the amnioserosa andperipheral nervous system. A number of different strategies can beemployed to obtain the integration of the transgene with the requisiteexpression pattern. Generally, methods of producing the subjecttransgenic flies involve stable integration of the transgene into thefly genome. Stable integration is achieved by first introducing thetransgene into a cell or cells of the fly, e.g. a fly embryo. Thetransgene is generally present on a suitable vector, such as a plasmid.Transgene introduction may be accomplished using any convenientprotocol, where suitable protocols include: electroporation,microinjection, vesicle delivery, e.g. liposome delivery vehicles, andthe like. Following introduction of the transgene into the cell(s), thetransgene is stably integrated into the genome of the cell. Stableintegration may be either site specific or random, but is generallyrandom.

Where integration is random, the transgene is typically integrated withthe use of transposase. In such embodiments, the transgene is introducedinto the cell(s) within a vector that includes the requisite P element,terminal 31 base pair inverted repeats. Where the cell into which thetransgene is to be integrated does not comprise an endogenoustransposase, a vector encoding a transposase is also introduced into thecell, e.g. a helper plasmid comprising a transposase gene, such aspTURBO (as disclosed in Steller & Pirrotta, “P Transposons Controlled bythe Heat Shock Promoter,” Mol. Cell. Biol. (1986) 6:1640-1649). Methodsof random integration of transgenes into the genome of a targetDrosophila melanogaster cell(s) are disclosed in U.S. Pat. No.4,670,388, the disclosure of which is herein incorporated by reference.

In those embodiments in which the transgene is stably integrated in arandom fashion into the fly genome, means are also provided forselectively expressing the transgene at the appropriate time duringdevelopment of the fly. In other words, means are provided for obtainingtargeted expression of the transgene. To obtain the desired targetedexpression of the randomly integrated transgene, integration ofparticular promoter upstream of the transgene, as a single unit in the Pelement vector may be employed. Alternatively, a transactivator thatmediates expression of the transgene may be employed. Of particularinterest is the GAL4 system described in Brand & Perrimon, supra.

In this particular embodiment, the subject transgenic flies are producedby: (1) generating two separate lines of transgenic flies: (a) a firstline that expresses GAL4 in mainly the amnioserosa and peripheralnervous system, e.g. under the control the endogenous fly promoterlocated on the 4^(th) chromosome (as found in fly line #3734 describedsupra); and (b) a second line in which the transgene is stablyintegrated into the cell genome and is fused to a UAS domain; (2)crossing the two lines; and (3) screening the progeny for the desiredphenotype, i.e. spontaneous development of neoplastic tumors. Each ofthe above steps are well known to those of skill in the art. See e.g.Brand & Perrimon, Development (1993) 118: 401-415; and Phelps & Brand,Methods (April 1998) 14:367-379. See also the Experimental Section,infra.

The above strategy is employed to obtain fertilized eggs that comprisethe transgene stably integrated into the genome in a manner such that itis expressed in the correct spatial and temporal manner so that the eggsgive rise to adult flies exhibiting the desired neoplastic phenotype.Generally, the fertilized eggs are allowed to mature under conditionsthat give rise to the neoplastic phenotype.

The neoplastic phenotype of the animals can be tailored by varying theconditions under which the animals are allowed to mature. For example,temperature can be employed to modulate the nature neoplastic phenotypeof the subject animals. Rearing the embryos/larva at 30-31° C. inducesmetastatic tumors in all larva, while rearing at 28° C. results in fewlarva generate metastatic tumors. Rearing at 22-24° C. results in themajority of the larva having metastatic tumors. Finally, growth at 18°C. results in few larva that generate tumors, but the larva that dogenerate tumors have extremely metastatic tumors.

Utility

The subject flies find use in a variety of applications, including: astools for use in the elucidation of genetic mechanisms involved incellular proliferative disorders; as a screening tool that identifiestherapeutic compounds for use in the treatment of cellular proliferativeconditions (e.g. as animal models for human neoplastic diseaseconditions); and as tools for use in the identification neoplastic genetargets, i.e. genes whose expression can be modulated, e.g. enhanced ordisrupted, in order to alleviate a neoplastic condition. The subjecttransgenic flies find particular use in screening methods designed toidentify therapeutic agents for use in the treatment of neoplasticdiseases.

Screening Methods

As mentioned above, the subject transgenic flies find particular utilityin screening assays designed to identify therapeutic compounds forcellular proliferative conditions, particularly neoplastic conditions.Through use of the subject transgenic flies (or cells derived therefromdepending on the particular screening assay), one can identify compoundsthat have activity with respect to a neoplastic disease. Compounds haveactivity with respect to a neoplastic disease if they modulate or havean effect on at least one parameter or symptom of the disease, such asloss abnormal cell division or complications associated therewith, wherethe modulatory activity may be to reduce or enhance the magnitude of thesymptom, depending on the nature of the disease and the symptom. Thus,the screening methods of subject invention can be used to identifycompounds that modulate the progression of neoplastic diseases, e.g. bybinding to, modulating, enhancing or repressing the activity of aprotein or peptide involved in the progression of the neoplasticdisease, and/or compounds that ameliorate, alleviate or even remove thephenotypic symptoms of the disease, where such activity may or may notbe the result of activity with respect to the underlying mechanism ofthe disease. Screening to determine drugs that lack effect on theneoplastic condition is also of interest. Assays of the invention makeit possible to identify compounds which ultimately: (1) have a positiveaffect with respect to a neoplastic disease condition and as such aretherapeutics, e.g. agents which arrest or reverse the neoplasticcondition or ameliorate or alleviate the symptoms of such a condition;or (2) have an adverse affect with respect to the neoplastic disease andas such should be avoided as therapeutic agents.

In the screening methods of the subject invention, a quantity of acandidate agent is generally orally administered to the fly. Followingoral administration, the affect of the candidate agent on the metastatictumors (or appearance thereof) of the fly is determined, typically bycomparison with a control (i.e. a transgenic fly to which the candidateagent has not been administered). The affect of the candidate agent isdetermined by determining whether one or more of the phenotypiccharacteristics of the neoplastic condition are exacerbated orameliorated in the test fly as compared to the control fly, wherecharacteristics that are monitored include tumor growth, tumormetastasis, and the like. The candidate agent is generally orallyadministered to the fly by mixing the agent into the fly nutrientmedium, e.g. water, aqueous solution with additional nutrient agents,etc., and placing the medium in the presence of the fly, (either thelarva or adult fly, usually the adult fly) such that the fly feeds onthe medium. Generally a plurality of assay mixtures are run in parallelwith different agent concentrations to obtain a differential response tothe various concentrations of candidate agent. Typically, one of theseconcentrations serves as a negative control, i.e. no compound. In apreferred embodiment, a high throughput screening protocol is employed,in which a large number of candidate agents are tested in parallel usinga large number of flies. By “large number” is meant a plurality, whereplurality means at least 10 to 50, usually at least 100, and moreusually at least 1000, where the number of may be 10,000 or 50,000 ormore, but in many instances will not exceed 5000.

Of particularinterest in certain embodiments is the use of the subjectflies in a high throughput toxicity screening assays, as described inU.S. patent application Ser. No. 60/147,220 filed Aug. 4, 1999, thedisclosure of which is herein incorporated by reference. In such highthroughput screening assays, a plurality of different compoundcompositions, usually at least 10 different compound compositions, aresimultaneously assayed for their toxic activity, if any. Each compoundcomposition in the plurality is assayed for toxicity by contacting itwith a population of the subject transgenic animals having a neoplasticphenotype and determining the effect of the compound composition on theanimals. Such HTS methods find particular use in finding agents for usein the treatment of cellular proliferative diseases, e.g. neoplasticdiseases, as only those compounds that treat the disease and yet aresufficiently non-toxic to allow the animal to live are identified aspositives for further study.

The subject methods find use in the screening of a variety of differentpotentially therapeutic candidate agents. Candidate agents encompassnumerous chemical classes, though typically they are organic molecules,preferably small organic compounds having a molecular weight of morethan 50 and less than about 2,500 daltons. Candidate agents comprisefunctional groups necessary for structural interaction with proteins,particularly hydrogen bonding, and typically include at least an amine,carbonyl, hydroxyl or carboxyl group, preferably at least two of thefunctional chemical groups. The candidate agents often comprise cyclicalcarbon or heterocyclic structures and/or aromatic or polyaromaticstructures substituted with one or more of the above functional groups.Candidate agents are also found among biomolecules including, but notlimited to: peptides, saccharides, fatty acids, steroids, purines,pyrimidines, derivatives, structural analogs or combinations thereof.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides and oligopeptides. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsare available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs. New potential therapeutic agents may also be createdusing methods such as rational drug design or computer modelling.

Screening may be directed to known pharmacologically active compoundsand chemical analogs thereof, or to new agents with unknown propertiessuch as those created through rational drug design. Candidate agentshaving therapeutic activity with respect to the neoplastic condition canbe identified based on their ability to at least ameliorate, if notcompletely alleviate or remove, one or more of the neoplastic phenotypesof the adult transgenic fly of the subject invention, such as tumorgrowth, tumor metastasis, and the like, as described above.

Of particular interest is the use of the subject screening methods toidentify cancer therapeutic agents that exhibit low host toxicity andyet are effective as antineoplastic agents. Tradition chemotherapy andradiation treatments affect normal and tumor cells alike. The tolerancefor normal cells is high because the primary mechanism of thesetreatments affect only dividing cells and since the majority of normalcells are not dividing the side effects are tolerated, but still quitesevere. The subject screening methods place a high stringency on sideeffects as a significant amount of cell division is required for thelarva to develop into a viable fly. Therefore the anti-metastatic tumorcompounds selected by the subject screening methods will have to behighly specific to kill or inhibit tumor formation/growth/metastisis,otherwise the developing fly will die. In other words, the subjectmethods represent a sensitive system for selecting anti-tumor drugcandidates that also have low toxicity to normal developing and dividingcells.

The above screening methods may be part of a multi-step screeningprocess of evaluating candidate therapeutic agents for their efficacy(and safety) in the treatment of neoplastic diseases in mammalian hosts,e.g. humans. In multi-step screening processes of the subject invention,a candidate compound or library of compounds is subjected to screeningin a second in vivo model, e.g. a mouse model, following screening inthe subject transgenic animal model. Following the initial screening inthe non-mammalian transgenic animals of the subject invention, thepositive compounds are then screened in non-human mammalian animalmodels, including transgenic non-human mammalian animal models.Transgenic mouse models of neoplastic diseases and methods for their usein screening assays are described in: U.S. Pat. Nos. 5,917,124;5,907,078; 5,849,996; 5,709,844; 5,550,316; and 4,736,866, thedisclosures of which are herein incorporated by reference. In addition,a pre in vivo screening step may be employed, in which the compound isfirst subjected to an in vitro screening assay for its potential as atherapeutic agent in the treatment neoplastic conditions. Any convenientin vitro screening assay may be employed, where a variety of suitable invitro screening assays are known to those of skill in the art.

Identification of Gene Targets

In addition to their use as animal models for screening candidatetherapeutic agents, the subject transgenic flies also find use in theidentification of neoplastic gene targets, i.e. genes whose expressioncan be beneficially modulated to treat neoplastic diseases. Gene basedtherapies can be identified by doing traditional enhancer/suppressoranalyses in the subject transgenic flies. In these analyses, genes inthe subject transgenic flies are mutated to identify ones that eitherexacerbate or alleviate the neoplastic phenotype. Methods of mutatinggenes and carrying out enhancer/suppressor analyses are well known tothose of skill in the art (Hays, T S et al., Molecular and CellularBiology (March 1989) 9(3):875-84; Deuring, R; Robertson, B; Prout, M;and Fuller, Mont. Mol. Cell. Biol., 1989 9:875-84.; Fuller, Mont. etal., Cell Mot. Cyto. (1989) 14:128-35; Rottgen G, Wagner T, Hinz U Mol.Gen. Genet. 1998 257:442-51).

Genes that mutate to enhance the neoplastic phenotype in a recessivemanner yield potential protein therapeutics for neoplastic conditions,since elevating the normal gene product level of such genes potentiallyalleviates the neoplastic condition. Genes that mutate to suppress theneoplastic condition in a recessive manner yield gene targets fordisruption to alleviate the neoplastic conditions, where disruption ofthese genes can be achieved using a variety of methods, ranging fromdeleting the DNA for the target gene to inhibiting its transcription,translation, or protein activity. For screening candidate agents, smallmolecule antagonists to these genes can be constructed and evaluated forefficacy in the fly model through oral administration. Alternatively,large molecular antagonists can be delivered by gene therapy, asdescribed infra.

Kits

Also provided by the subject invention are kits for use in performingthe subject screening methods. The subject kits include at a pluralityof transgenic flies of the subject invention, or a means for producingsuch a plurality of flies, e.g. a male and female transgenic fly of thesubject invention, vectors carrying requisite genes, such as thetransgene, a transposase gene, GAL4, etc. The flies may be housed inappropriate container(s), e.g. vials. The subject kits may also comprisea nutrient medium for the animals, e.g. drosophila medium.

Therapeutic Agents and Pharmaceutical Compositions

Also provided by the subject invention are therapeutic agents for use intreating a neoplastic condition, as well as pharmaceutical formulationsthereof. The therapeutic agents of the subject invention are thoseagents identified using the screening methods described supra that showbeneficial activity with respect to a neoplastic condition (or agentsknown to have an effect on the expression of a gene identified asmodulating the phenotype of a neoplastic condition, where identificationemploys the use of the subject non-transgenic animals).

Also provided are pharmaceutical compositions of the subject therapeuticagents. In the pharmaceutical compositions or formulations of thesubject invention, agents described above are formulated intopharmaceutical compositions by combination with appropriate,pharmaceutically acceptable carriers or diluents, and may be formulatedinto preparations in solid, semi-solid, liquid or gaseous forms, such astablets, capsules, powders, granules, ointments, solutions,suppositories, injections, inhalants and aerosols. In pharmaceuticaldosage forms, the agents may be administered in the form of theirpharmaceutically acceptable salts, or they may also be used alone or inappropriate association, as well as in combination, with otherpharmaceutically active compounds. The following methods and excipientsare merely exemplary and are in no way limiting.

For oral preparations, the agents can be used alone or in combinationwith appropriate additives to make tablets, powders, granules orcapsules, for example, with conventional additives, such as lactose,mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

The agents can be formulated into preparations for injection bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives.

The agents can be utilized in aerosol formulation to be administered viainhalation. The compounds of the present invention can be formulatedinto pressurized acceptable propellants such as dichlorodifluoromethane,propane, nitrogen and the like.

Furthermore, the agents can be made into suppositories by mixing with avariety of bases such as emulsifying bases or water-soluble bases. Thecompounds of the present invention can be administered rectally via asuppository. The suppository can include vehicles such as cocoa butter,carbowaxes and polyethylene glycols, which melt at body temperature, yetare solidified at room temperature.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, tablespoonful, tablet or suppository, contains a predeterminedamount of the composition containing one or more inhibitors. Similarly,unit dosage forms for injection or intravenous administration maycomprise the inhibitor(s) in a composition as a solution in sterilewater, normal saline or another pharmaceutically acceptable carrier.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present invention calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the novel unitdosage forms of the present invention depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with each compound in the host.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

Where the agent is a polypeptide, polynucleotide, analog or mimeticthereof (as identified using the mutant screen analysis protocolsdescribed supra, it may be introduced into tissues or host cells by anynumber of routes, including viral infection, microinjection, or fusionof vesicles. Jet injection may also be used for intramuscularadministration, as described by Furth et al. (1992), Anal Biochem205:365-368. The DNA may be coated onto gold microparticles, anddelivered intradermally by a particle bombardment device, or “gene gun”as described in the literature (see, for example, Tang et al. (1992),Nature 356:152-154), where gold microprojectiles are coated with theDNA, then bombarded into skin cells.

Those of skill in the art will readily appreciate that dose levels canvary as a function of the specific compound, the severity of thesymptoms and the susceptibility of the subject to side effects.Preferred dosages for a given compound are readily determinable by thoseof skill in the art by a variety of means.

Kits with unit doses of the active agent, usually in oral or injectabledoses, are provided. In such kits, in addition to the containerscontaining the unit doses will be an informational package insertdescribing the use and attendant benefits of the drugs in treatingpathological condition of interest. Preferred compounds and unit dosesare those described herein above.

Methods of Treating Neoplastic Conditions

Also provided are methods of treating cellular proliferative diseaseconditions, particularly neoplastic disease conditions, using thesubject active agents. In the subject methods, an effective amount ofthe active agent of the subject invention is administered to the host tobe treated. By “effective amount” is meant a dosage sufficient toproduce a desired result, where the desired result is generally anamelioration or alleviation, if not complete cessation, of one or moresymptoms of the cellular proliferative disease being treated.Administration of the agents can be achieved in various ways, includingoral, buccal, rectal, parenteral, intraperitoneal, intradermal,transdermal, intracheal, etc. A variety of hosts are treatable accordingto the subject methods. Generally such hosts are “mammals” or“mammalian,” where these terms are used broadly to describe organismswhich are within the class mammalia, including the orders carnivore(e.g., dogs and cats), rodentia (e.g., muce, guinea pigs, and rats), andprimates (e.g., humans, chimpanzees, and monkeys). In many embodiments,the hosts will be humans.

The following examples are offered by way of illustration and not by wayof limitation.

Experimental

A. Preparation of Flies Containing the pUAST-v-myb-1151 Construct

The v-m yb-1151 gene (as described in Fu and Lipsick, 1996, supra) wascloned into the polylinker of the pUAST such that the UAS vectorsequences were adjacent to the 5′ end of the myb gene (Brand et al.1993, supra). Transcription of the construct was controlled by GAL4,which needs to bind to the UAS sequence to drive expression of the genethat has been cloned into the pUAST vector. The pUAST-v-myb-1151construct was integrated into the genome of Drosophila melanogaster bystandard microinjection procedures (Spradling, A. C., and Rubin, G. M.(1982). Science 218, 341-347). The transposase source used to stimulatethe integration of the pUAST-myb-1151 construct into the genome wasprovided by co-injecting the vector pTURBO (as described in Steller &Pirrotta, (1986) supra).

B. Production of Flies Having a Neoplastic Phenotype

Flies containing the pUAST-v-myb-1151 construct were mated to the #3734GAL4 producing fly strain (as described in Brand (1993) supra) andobtained from the Bloomington stock center in Indiana(http://flybase.bio.indiana.edu/) and their progeny were allowed todevelop. TABLE 1 Phenotypes of Tumor Afflicated Larva over a TemperatureRange Temperature % of larva with % of larva with (° C.) 1+ tumors 2+tumors 18  6 6 22 70 58  28 36 4 30-31 100  100 Larva = third instar larvaThe number scored for each temperature = 50

The resultant flies spontaneously produced tumors. See FIGS. 1A to 1C.Tumors (dark colored, dense clusters of dividing cells) were dissectedfrom the animals and their growth was examined in culture. Standardinsect cell culture media was used (Sigma-Aldrich) and cells wereincubated at 22° C. The volume of the tumor doubled in 5 to 7 days.Furthermore, our studies suggest that normal larva are killed byreceiving a tumor transplant. These results indicate that these areclassical tumors that are being generated in the v-myb expressing fruitfly. Transplantation of tumors into normal larva viable flies MockSurgury 33.3% Tumor Surgury  7.8%The number of scorable mock surguries performed=54The number of scorable tumor suguries performed=51

A scorable surgery is one where post operation the larva moves normallyand no internal fluid leaks out from the incision.

C. Screening Assays

A variety of herbs and Chinese medicines have been identified for havingantitumor activity. Those in Table 2 have activity against a variety ofcancer types, which includes: hepatic, colon, leukemia, lymphoma,glioma, breast, prostate, pancreas, bladder, melanoma, and lung. Seee.g. Zheng, et al. Immunopharmacol Immunotoxicol 1995. 17: 69-79;Borchers, et al., Proc. Soc. Exp. Biol. Med. 1999. 221: 281-293; Hu, etal., Planta Med 1996. 62: 573-575; Zheng, et al., J. Cell Biochem.Suppl. 1997. 27: 106-112; Nakahata, et al., J. Chin. Med. 1998. 26:311-23; Kato, et al., J. Invest. Dermatol. 1998. 111: 640-644; Huang, etal., J. Med. 1997. 46:132-137;Yamashiki, et al., J. Gastroenterol.Hepatol. 1996. 11: 137-142; Sakamoto, et al. Am. J. Chin. Med. 1994. 22:43-50; Ito & Shimura, Jpn. J. Pharmacol. 1986. 41: 307-314; Michaud, etal. J. Natl. Cancer Inst. 1999. 91: 605-613; Sasaki, et al. Nutr. Cancer1999. 33: 76-81; Sengupta & Das, Eur. J. Cancer Prev. 1999. 8: 325-330.The extract concentration fed to the larva/fly that delayed tumorprogression by 20% or more is indicated in Table 2. Furthermore, drugstreating this condition appear to be specific for antitumor propertiesas random extracts fed to the flies identify a positive ˜0.0025% of thetime. TABLE 2 Extracts of herbs and Chinese medicines that are known tohave antitumor activity in mammals show a similar activity in our tumorfly model. Extract tested for antitumor activity Positive antitumoractivity dose range Ginkgo biloba leaf .01%-100% Trichosanthes  10%-100%Grifola  10%-100% Dioscorea  .01%-.1%  Rhizoma zedoariae   1%-100%Broccoli .01%-100% Lycopene .01%-100% Sho Saiko to  .01%-.1% 

The ability of known chemicals to affect the tumors in this mutant flystrain indicates that it is a capable of identifying chemicals that willbe beneficial for treating human cancers. As the tumors in this mutantfly strain are affected by chemicals that target a variety of tumortypes in mammals, these fly tumors are a primitive generic tumor. Thatis, this tumor has the basic properties of cancer, however lacking themore sophisticated mechanisms associated with specific organ/tissues. inmammals. For example, flies do not possess breast or pancreas tissues.However, these tumors are inhibited by chemicals that target breast andpancreas tumor types. As such, at least some of the fundamentalproperties are the same in both the fly tumors as well as pancreas andbreast cancers. Therefore, the subject flies have uncovered tumorprogression pathways that are common for most cancers.

It is evident from the above results and discussion that the subjectinvention provides a valuable screening tool for use in the evaluationof potential therapeutic agents for use in the treatment of cellularproliferative disorders. Advantages of using the subject transgenicflies for screening potential therapeutic candidates include:adaptability of the subject flies to high throughput screeningprotocols, simplicity and low cost of maintaining the subject flies,ability of the subject flies to identify potentially orally activetherapeutic agents, rapid reproduction of the subject flies, and abilityof the subject flies to produce large numbers of offspring. Accordingly,the subject invention fills a void in the existing arsenal of screeningtools, in that the subject invention provides a means for conducting invivo high throughput screening assays. A further significant advantageis the ability to use the subject flies to identify compounds thatexhibit low or no toxicity to normal dividing cells but still exhibitsufficient toxicity to abnormally dividing cells. As such, the subjectscreening methods provide a means for identifying effectiveanti-neoplastic agents that exhibit low or no toxicity to normal cells.Therefore, the subject invention represents a significant contributionto the art.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

1. A non-mammalian transgenic animal, wherein said transgenic animalspontaneously develops metastatic tumors and comprises a v-mybtransgene.
 2. The transgenic non-mammalian animal according to claim 1,wherein said transgenic non-mammalian animal is an invertebrate.
 3. Thetransgenic non-mammalian animal according to claim 2, wherein saidinvertebrate is an insect.
 4. A transgenic fly that spontaneouslydevelops metastatic tumors and comprises a v-myb transgene.
 5. Thetransgenic fly according to claim 4, wherein said fly is a member of thefamily Drosophilidae.
 6. The transgenic fly according to claim 5,wherein said fly is a Drosophila melanogaster.
 7. The transgenic flyaccording to claim 4, wherein said v-myb transgene is expressed in theamnioserosa and peripheral nervous system.
 8. A transgenic Drosophilamelanogaster that spontaneously develops metastatic tumors and comprisesa v-myb transgene.
 9. A method of screening a compound for activity withrespect to cellular proliferative diseases, said method comprising:administering said compound to a non-mammalian animal according to claim1; and observing the effect of said compound on said animal.
 10. Themethod according to claim 9, wherein said non-mammalian animal is atransgenic invertebrate.
 11. The method according to claim 10, whereinsaid invertebrate is an insect.
 12. The method according to claim 11,wherein said insect is a fly.
 13. The method according to claim 9,wherein said compound is orally administered to said animal.
 14. Amethod of screening a compound for activity with respect to a neoplasticdisease, said method comprising: feeding said compound to anon-mammalian animal according to claim 1; and observing the effect ofsaid compound on said animal.
 15. The method according to claim 14,wherein said compound is present in a nutrient medium.
 16. A method ofscreening a plurality of compounds for activity with respect to cellularproliferative disease, said method comprising: feeding said plurality ofcompounds to a plurality of non-mammalian animals according to claim 1in a manner sufficient to ensure that each animal is fed only a singletype compound from said plurality of compounds; and observing the effectof said compounds on said plurality of animals.
 17. The method accordingto claim 16, wherein said compound is present in a nutrient medium.(nutrient medium includes water)
 18. A method of identifying whether agene is capable of modulating a neoplastic disease condition, saidmethod comprising: mutating said gene in a non-mammalian animalaccording to claim 1; and determining whether said mutation modulatesthe phenotype of said non-mammalian animal model.
 19. The methodaccording to claim 18, wherein said modulating is enhancing.
 20. Themethod according to claim 18, wherein said modulating is suppressing.